Osmotic stress from saline environments is experienced by a wide range of organisms in nature. For example, bacteria expelled into the ocean in sewage and higher plants coping with drying fields must dramatically increase their intracellular osmotic pressure to maintain cellular turgor pressure. Similar osmotic pressure gradients are created in the kidneys of higher animals. There is increasing evidence that these diverse organisms use analogous strategies to cope with the stress. The goal of this research program is to contribute to understanding the molecular mechanisms of adaptation to environmental stress by elucidating the mechanism of osmoregulation; the altered pattern of gene expression brought about by a change in environmental osmotic strength. Escherichia coli's growth and survival in saline environments is greatly enhanced by "osmoprotectants"; small organic molecules that can be accumulated to high internal concentrations without disturbing cellular metabolism. A transport system for the ubiquitous osmoprotectant, glycine betaine, is encoded by the proU operon. This project will analyze the mechanism of induction of the genes encoding the transport system by elevated osmolarity. The role of the osmotically inducible osmB and osmD genes in the adaptative response will be determined by identifying their protein products and functions. A genetic search for regulatory loci will be carried out. The possibility of coordinate regulation of osmotically inducible genes, and common regulatory signals will be examined. The chemical nature of the osmoregulatory signal will be explored in permeabilized cells, in vitro coupled transcription/translation and reconstituted transcription. The technical emphasis is on biochemical analysis of components of the osmotic response, with genetic manipulations aiding and supporting this effort.